Healing of serious diseases in vivo is often limited by the inability of the body to rebuild a sufficient amount of healthy cells. Especially in neurons and chondrocytes (cartilaginous tissue) this is a big challenge. One research goal of many stem cell facilities is to understand the molecular mechanisms of diseases like e.g. Parkinson’s disease (PD) or to create models for reconstructive medicine and stem cell therapies. For these scientists, induced pluripotent stem (iPS) cells are a highly valuable tool in their research.
Stem Cells (IPSc + ESc) As Pluripotent Possibility Of Science
Stem cells have the ability to differentiate into nearly all cell types of the organism. A distinction is made between embryonic stem cells (ES cells) and induced pluripotent cells (iPS cells). Since a decade, especially the iPS cells came more and more into the focus of stem cell research.
The main work in this field of research consists of iPS cell derivation, cultivation, genetic modification, characterization of pluripotency of ES and iPS cells and finally the differentiation of this cell type.
Non-Invasive iPS Cell Detection – Gentle And Save Stem Cell Techniques
[iPS Colony Count]
Since iPS cells are very rare and of high value, a reliable and highly sensitive measuring method for the success of the studies is essential.
CELLAVISTA® and NYONE® in combination with the YT-Image Analysis Software® are capable of detecting iPS colonies grown on feeder layer. The challenge is to identify these colonies which appear in a weak brightfield contrast image during the early stage of growth.
To differentiate human iPS cells, these cells were seeded on a special feeder cell layer. After a defined growth period the size and number of the in the 2nd dimension grown iPS colonies can fast be determined by brightfield measurement with the 4x magnification.
Extract from the software readout for adherent cells (for further information look at ShortNote iPS Colony Count)
- # of iPS Colonies
- Cell Confluence [%]
- iPS Colony Area [%]
- average Colony Size [µm2]
Fluorescent Modification Of Stem Cell Proteins – A Very Efficient Method
[Virtual Cytoplasm (1F)]
The investigation of induced pluripotent stem (iPS) cells is moving more and more into the focus of research! Different systems to generate cell sources which produce hyaline cartilage with the aim to repair cartilage injury in future are still developed.
Different reprogramming methods to generate chondrocytes using fibroblasts are under investigation: e.g. direct reprogramming of fibroblasts into chondrocytes (iChon cells) and the generation of chondrocytes from reprogramming adult dermal fibroblasts to an undifferentiated state (Hiramatsu et. al 2011).
During their studies the CELLAVISTA® with its high resolution, strong detection capabilities and advanced autofocus functionality has strongly supported their need to identify cells expressing low GFP signals as marker for induced cell properties.
Extract from the software readout for adherent cells (for further information look at ShortNote Virtual Cytoplasm 1F)
- total # of Cells
- # of F1 Marker expressing Cells
- % of F1 marker expressing Cells
- Average Fluorescence Intensity
iPS Characterization – Using Extensive List Of Marker Proteins
[Confluence (dots 1F)]
The genetically modification of e.g. human embryonic stem cells (HESCs) or human induced pluripotent stem cells (hiPSCs) becomes an essential tool in studying stem cell biology and developing potential clinical approaches.
Despite the standard use of this method, it is associated with many difficulties. Everyone knows how challenging and time consuming it may be to establish an ideal transfection protocol. Even if the desired vector is designed and cloned, the way to a reliable transfection is still far!
A widely applied method is the expression of a selection marker in combination with a fluorescent marker gene (e.g. eGFP, DsRED, mCherry…) additionally to the target gene. The read out for the qualitative and quantitative analysis of transfection will be usually performed by user-dependent and time-consuming manual microscopy or, respectively, precise but invasive flow cytometry.
In contrast to these methods SYNENTEC’s cell imagers quantify the transfection efficiency and subsequently the quantification of the expression of the gene of interest within minutes via software assisted image analysis within a wide range of fluorescence spectra. Our YT-Software® can analyze a whole microplate non-invasively and even track your culture over days to determine growth-curves and and thereby the health of your stem cells.
Extract from the software readout for adherent cells (for further information look at ShortNote Confluence (dots 1F))
- % Confluence BF
- % Confluence FL
- FL Objects on BF Area/ BF Area
Recombinant Lectins As Probes For Undifferentiated hES and hiPS Cells
[Virtual Cytoplasm (1F) + (2F)]
New developed recombinant BC2LCN can be used for research of human induced pluripotent stem (iPS) and embryonic stem (ES) cells. Those recombinant lectins (e.g. rBC2LCN lectin) can be coupled with fluorescent marker, such as fluoroisothiocyanate (FITC) or rhodamine derivatives and are suitable as new probes for the localization of glycoconjugates on cell surfaces.
rBC2LCN is expected to be applicable not only to stem cell research but also to regenerative medicine since it is reportedly a useful marker for the detection of undifferentiated cells. rBC2LCN reportedly recognized exclusively an intramolecular sugar chain of Podocalyxin which exposes on the cell surface of undifferentiated hiPS and human ES.
CELLAVISTA® and NYONE® in combination with the YT-image analysis software® are capable to detect and evaluate the fluorescent labeled r-lectin-probes which specifically mark undifferentiated stem cells.
Extract from the software readout for adherent cells (for further information look at ShortNote Virtual Cytoplasm (2F))
- total # of Cells
- % of Hoechst33342 and rBC2LCN-Cy3 stained Cells or:
- % of Hoechst33342 and rBC2LCN-FITC stained Cells
Cell Proliferation – Keep An Eye On It
[Confluence – Suspension Cell Count]
Confluence monitoring is a useful tool to determine various properties of your cell line. SYNENTEC’s confluence image processing analysis is capable to solve a vast range of different questions – e.g. to monitor the growth rate under selective conditions or to prove effectiveness by adding a differentiation factor and monitor the growth rate over time as a first step of proving the influence on the cell type of interest.
With SYNENTEC’s automated cell culture microscopes NYONE® and CELLAVISTA® and the included YT-image analysis software® the proliferation analysis is possible for a variety of cells. Adherent lines can be detected as well as suspension cells. In addition, it is very easy to quantify additional fluorescent labels and their ratio to the total growth area.
Extract from the software readout for adherent cells (for further information look at ShortNote Confluence)
- covered Area [µm2]
- evaluated Area [µm2]
- Confluence [%]